The expression of interferon-lambda1 in CHO cell / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells.</p><p><b>METHODS</b>Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1.</p><p><b>RESULTS</b>Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity.</p><p><b>CONCLUSION</b>Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.</p>

Preparation of the monoclonal antibody against human Bocavirus VP2 protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express and purify HBoV VP2 protein, and the monoclonal antibody against HBoV VP2 protein was prepared with hybridoma technique.</p><p><b>METHODS</b>The HBoV VP2 cloned into vector pET-30a was expressed in E. coil. After purified by immobilized metal affinity chromatography, the BALB/c mouse was immunized with purified protein as antigen. The positive hybridoma cells were screened with hybridoma technique and ELISA assay. Isotype and titer of the monoclonal antibody were detected.</p><p><b>RESULTS</b>The recombinant HBoV VP2 protein was expressed and purified, and then the monoclonal antibody was obtained with hybridoma technique. The titer of the IgG monoclonal antibody was up to 1:4 x 10(5).</p><p><b>CONCLUSION</b>Monoclonal antibody against recombinant HBoV VP2 protein was prepared and the antibody titer was high. This work may provide a new method in rapid diagnosis and study of HBoV.</p>

Construction of human metapneumovirus DNA vaccine and study on its immune response in mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct human metapneumovirus (hMPV) DNA vaccines and evaluate the cellular and humoral immune response in mice.</p><p><b>METHODS</b>Fusion protein FdeltaTM (without transmembrane domain) gene and M gene of hMPV were amplified from cDNA by PCR, then DNA vaccines pcDNA3.1His-FdeltaTM and pcDNA3.1His-M were constructed to verify the expression of F and M protein by Western blotting and indirect immunofluorescent assay (IFA) respectively. Serum IgG and spleen cell CTL were detected with ELISA and ELISPOT assay after the BALB/c mice were immunized intramuscularly with the vaccines.</p><p><b>RESULTS</b>The candidate DNA vaccines could express FdeltaTM and M protein as detected with Western blotting and IFA. The IgG antibody titers of mice was 1:44 when immunized with pcDNA3.1His-FdeltaTM, but could increase to 1:64 when co-immunized with pcDNA3.1His-M. ELISPOT assay demonstrated that IFN-gamma-secreting effector T cells reached 42 +/- 8.9 in co-immunization group, higher than single vaccine pcDNA3.1His-FdeltaTM group (32 +/- 7.4).</p><p><b>CONCLUSION</b>DNA vaccine pcDNA3.1His-FdeltaTM could induce specific cellular and humoral immune responses, and the immune response could increase when co-immunization with pcDNA3.1His-M was carried out.</p>

Analysis of human metapneumovirus genotype in Hunan, China and its attachment protein G sequence character / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand the genotypes of human metapneumovirus (hMPV) and the genetic character of hMPV attachment protein G sequence in Hunan, China.</p><p><b>METHODS</b>232 nasopharyngeal aspirates (NPA) samples from hospitalized children with acute respiratory infections were collected from Hunan, China in 2005. HMPV was detected. The full length of G glycoprotein genes were amplified and sequenced. Bioinformatics soft-wares were employed to analyze the sequences.</p><p><b>RESULTS</b>17/232 (7.3%) were showed hMPV positive. And co-infection rate with other viruses is 35%. The diagnoses of these hMPV positive cases are pneumonia, bronchiolitis and bronchopneumonia. Phylogenetic analysis for G genes from 13 hMPVs revealed the existence of four major subgroups: A1, A2, B1, B2 in Hunan, China in 2005. There are four types of sequence lengths of hMPV G glycoprotein, which are 711, 675, 660, 696nt. It is different in potential N-linked glycosylation sites and number of cysteine residues among these hMPVs of Hunan, China and Beijing, China. Also it is different from those in Japan and North America.</p><p><b>CONCLUSION</b>The investigation of hMPV from Hunan, China in 2005 revealed the high speed of genetic variation and the marked character of geographic epidemic differences.</p>

WU polyomavirus and KI polyomavirus detected in specimens from children with acute respiratory tract infection in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate newly identified polyomavirus WUV and WUV and KIPyV are associated with acute respiratory infections in China, tests were developed to detect WUV and KIPyV gene fragments from nasopharyngeal aspirates collected from children with ARI fron Nov. 2006 to Oct. 2007.</p><p><b>METHODS</b>A total of 318 clinical samples were tested for WUV and KIPyV using PCR method. The positive products were sequenced and compared with those in GenBank.</p><p><b>RESULTS</b>14 of the 318 Samples were positive (WUV was 2.2%, KIPyV was 2.2%). All of children who were positive for WUV or KIPyV had respiratory illness.</p><p><b>CONCLUSION</b>Polyomavirus WU and KIPyV infection may be associated with upper and lower respiratory diseases.</p>

Genome cloning and phylogenetic analysis of human bocavirus capsid gene / 病毒学报

The full-length genome of one human bocavirus (HBoV) and the VP1 sequences of nine HBoV were amplified from patients' samples by PCR, cloned into pGEM-T vector separately, and sequenced. In this study, the one full length gemome and nine VP1 sequences of HBoV were aligened with 14 sequences of Parvoviruses which were canonical exemplars in Parvovirinae. Phylogenetic analysis showed that HBoV capsid sequences positioned closely to B19 parvovirus, although they positioned far in phylogenetic tree based on full length genome. Many similarities were found between HBoV and B19 in capsid by alignment on secondary structural elements. Because both B19 and HBoV are the only Parvoviruses that infect mankind, so study on HBoV may be used for reference to B19 which had been studied for about 30 years. By analysis of mutational sites, HBoV capsid protein showed a highly conserved secondary structural elements, but highly active in VP1-U, leading end of VP2 and insertions between the strands of the betaG-H. This cued that HBoV inclined to immune evasion and infectant adaptive faculty.

Expression, purification and antiviral activities of a new recombinant human interferon-lambda2 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express recombinant human interferon lambda2 in E.coli and to study its antiviral activities.</p><p><b>METHODS</b>According to preferred codons used in E.coli, the highly-expressed human interferon lambda2 gene was designed, synthesized and cloned into expression vector pBV220 and transfected into E.coli DH5alpha. The expressed product was purified by using CM FF and size exclusion chromatography. Its antiviral activities were tested on different cells.</p><p><b>RESULTS</b>The expressed product was calculated about 15% of the total E.coli protein. The purified protein reached about 90% purity. Its specific antiviral activity was about 1.5 x 10(6) IU/mg on WISH/VSV test system. It was shown that the antiviral activity of the product on primates-origin cells seemed to be much higher than that on other non-primates-origin cells, indicating that interferon lambda2 possessed more stringent species specificity as compared with interferon-alpha2b. New interferon lambda2 showed similar anti-HBV activity as interferon-alpha2b.</p><p><b>CONCLUSION</b>Recombinant human interferon lambda2 could be expressed on E.coli. The purified product showed more stringent species specificity and similar anti-HBV activity as compared with interferon-alpha2b.</p>

Rotavirus surveillance data from Kunming Children's Hospital, 1998 - 2001 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the epidemiological status on rotavirus diarrhea in Kunming to improve the rotavirus vaccine immunization program.</p><p><b>METHODS</b>A hospital-based sentinel surveillance program for rotavirus was set up among children less than 5 years old with acute diarrhea in Kunming Children's Hospital. Clinical information and fecal specimens were collected and rotavirus were detected by polyacrylamide gel electrophoresis (PAGE) and/or enzyme linked immunosorbent assay (ELISA). Positive specimens were further serotyped or genotyped by ELISA and/or RT-PCR.</p><p><b>RESULTS</b>During the three years of surveillance, 466 specimens were collected. Rotavirus were detected on 246 (52.8%) specimens. 97% of the rotavirus diarrhea cases occurred among children less than 2 years old. There was a peak of admissions for rotavirus diarrhea cases between October and December which accounted for 48% of all the rotavirus hospitalizations each year. Among 204 specimens with G serotyping, the predominant strain was serotype G1 (47.5%) followed by G2 (17.6%), G3 (15.7%), G9 (4.9%) and G4 (1.0%). Mixed infection (2.5%) were rare and 22 specimens (10.8%) remained non-typeable. P genotyping showed P[4], P[8] and P[6] were the most common strains, accounting for 29.3%, 27.6% and 13.8% respectively. P[4]G2 was the most common strain which accounted for 34.1% (14/41) followed by P[8]G1 (29.3%) and P[6]G9 (12.2%). Another 7 uncommon P-G combinations were also identified.</p><p><b>CONCLUSION</b>Rotavirus was the major cause of acute diarrhea in Kunming. An effective rotavirus vaccine for prevention and control of rotavirus diarrhea should be developed.</p>

Epidemiological study of human caliciviruses among children with acute diarrhea in Lulong county, 1999 - 2001 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the epidemiological characteristus of human caliciviruses (HuCVs) among children under 5 years of age with acute diarrhea and to estimate the disease burden in Lulong county.</p><p><b>METHODS</b>HuCVs were detected by enzyme linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Some PCR amplicons were cloned and sequenced. Phylogenetic tree was constructed for strain characterization. The rate of HuCVs-attributed hospitalization was estimated according to the positive rate of HuCVs detection in fecal specimens collected from hospitalized diarrhea patients.</p><p><b>RESULTS</b>Between July 1999 and June 2001, 708 fecal specimens were collected, of which 393 rotavirus-negative and 5 rotavirus-positive specimens were detected for HuCVs. Thirty-one point six percentage of fecal specimens from patients with diarrhea was HuCVs positive. Among inpatients, HuCVs positive rate was 17.5%. HuCVs detection was mainly distributed in 3 - 17 mouth-old children, in winter. All 11 strains belonged to NLV GII in which 6 strains GII-3, 2 strains GII-4 and 3 strains GII-7, and they shared 55.1% - 100% nucleotide identity. NLV GII-4 and GII-7 were identified in 2000, while NLV GII-3 and GII-7 in 2001. The preliminary estimate of HuCVs-attributed hospitalization rate was 3.6 per thousand.</p><p><b>CONCLUSION</b>Human caliciviruses with different genotypes circulated among children in Lulong county with GII NLVs were the prevalent strains. The disease burden of HuCVs was second to rotavirus.</p>

Epidemiological study of rotavirus diarrhea in Beijing, China - a hospital-based surveillance from 1998 - 2001 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To provide information on epidemiology of rotavirus infection in Beijing, China.</p><p><b>METHODS</b>An ongoing hospital-based surveillance was conducted among children < 5yr old with acute diarrhea according to WHO generic protocol (CID-98). During a 3-year study (Apr. 1998 to Mar. 2001), a total of 484 stool samples were collected from 1 457 patients, including 275 samples from 1 048 outpatients and 209 samples from 409 inpatients.</p><p><b>RESULTS</b>The overall detection rate of rotavirus infection was 25.4%. Rotavirus was responsible for 27.3% of diarrhea inpatients on a yearly base, and 46.2% during rotavirus season. Two peaks of diarrhea were observed each year, one in the summer (June-Sep.) due to bacterial dysentery (16.7%) and another in fall winter (Oct.-Dec.) due to rotavirus infection (23.0%). The detection rate on rotavirus was the highest in age group of 6 - 11 months (38.2%), followed by 1 - 2 years old (28.5%). Ninety six point eight percentage of children were infected under 3 years of age. The number of deaths, possibly caused by rotavirus diarrhea were accounted for 40% of all diarrhea deaths and 11.1% of the total deaths. Serotyping of 123 rotavirus isolates showed that serotype G1 (55.3%) was predominant, followed by G2 (26.8%), G3 (9.8%), G4 (0.8%), and 10 isolates (8.1%) remained non-typeable. Mixed infections (0.8%) seemed to be rare.</p><p><b>CONCLUSION</b>Rotavirus diarrhea was an important infectious disease among children in Beijing. Safe and effective rotavirus vaccines for the prevention of severe diarrheas and the reduction of treatment costs are of significant importance to China.</p>

Surveillance finding on rotavirus in Changchun children's hospital during July 1998-June 2001 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To establish baseline patterns of rotavirus diarrhea and to describe its epidemiologic features in Changchun city, prior to rotavirus vaccine immunization.</p><p><b>METHODS</b>Hospital-based surveillance was conducted among children under 5 years old with acute diarrhea in Changchun Children's Hospital. Fecal samples were determined to identify rotavirus by PAGE and/or ELISA. G serotypes of rotavirus were identified by ELISA and/or nested RT-PCR. P genotyping were carried out by RT-PCR. All data were computerized and analysed by "Generic Manual on Rotavirus Surveillance" set by CDC in the USA.</p><p><b>RESULTS</b>In total, 2 343 diarrhea cases were screened and 1 211 fecal samples were collected. Rotavirus was detected in 31.0% among outpatients and 52.9% in inpatients. During the peak of the season (November through March), 58.6% of diarrhea was caused by rotavirus among inpatients. 95.0% of rotavirus diarrhea cases occurred among children aged < 2 years. The predominant strain was serotype G1 (82.4%), followed by G2 (5.0%), G3 (3.3%), G4 (0.9%). P genotyping showed that P[8] and P[4] were the most common ones. Nine different P-G combinations were identified, four strains (P[8]G1, P[4]G2, P[8]G3, and P[8]G4) commonly seen worldwide accounted for 75.6% of the total. Taken together with uncommon strains, including the novel types P[4]G4 and P[8]G2, it highlights the extraordinary diversity of rotaviruses circulating in China.</p><p><b>CONCLUSION</b>Rotavirus is the major cause of severe child diarrhea in Changchun. Developing a rotavirus vaccine for prevention of severe disease and reduction of treatment costs seemed to be necessary.</p>

Investigation of an outbreak of acute diarrhea caused by Human calicivirus / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To survey a diarrhea outbreak in Guangan city and analyze the cause of the disease.</p><p><b>METHODS</b>The population enrolled in the surveillance came from four different settings and was randomly sampled. Stool specimens collected from diarrhea patients were tested ordinarily for enteric bacteria and further examined for viral pathogens with PAGE, ELISA and RT-PCR.</p><p><b>RESULTS</b>In total, 4,567 persons were surveyed, among them 942 had acute diarrhea (prevalence 20.63%). The incidence was higher in rural area (28.6%) than in urban area (19.6%) (chi-square =22.29, P less than 0.005) with a peak in May 10 through 25 four human caliciviruses were detected from stool specimens by ELISA and RT-PCR in specimens from 4 and 1 patients, respectively.</p><p><b>CONCLUSION</b>Human calicivirus probably was the cause of this diarrhea outbreak in Guangan city.</p>